Cell cycle-specific loading of condensin I is regulated by the N-terminal tail of its kleisin subunit

Hirano, Takeshi; Tane, Shoji; Shintomi, Keishi; Kinoshita, Kazuhisa; Tsubota, Yuko; Nishiyama, Tomoko

doi:10.1101/2022.08.19.504508

Cited by 2 publications

(3 citation statements)

References 42 publications

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“…To this end, the C-terminal IDR of XCAP-D2 (referred to as XD2-C WT) was expressed as a polypeptide fused to the maltose-binding protein, purified and incubated with M-CDK as a substrate. We found that M-CDK efficiently phosphorylated this fusion protein, causing mobility shifts on a Phos-tag gel Our recent work has shown that several SP and TP motifs in a recombinant mammalian condensin I complex are phosphorylated in mitotic egg extracts [30]. We confirmed that our M-CDK can phosphorylate at least two of them (T1339 and T1353 of human CAP-D2) in vitro…”

Section: Suc1 Accelerates M-cdk Phosphorylation Of a Substrate Contai...supporting

confidence: 78%

“…Typically, 20 µg of the protein complex was obtained from 1 g insect cell pellet at a concentration of 2 µM. A mammalian version of the condensin I holocomplex (composed of mouse Smc2 and Smc4, and human CAP-D2, -G, and -H) was prepared as previously described [29,30].…”

Section: Suc1 a Cdna Encoding Schizosaccharomyces Pombe Suc1 (Provide...mentioning

confidence: 99%

“…AfR16) [23]; anti-XCAP-H (in-house identifier: AfR20) [23]; anti-pT1314-XCAP-D2 (in-house identifier: AfR42-P) [24]; anti-pT1353-XCAP-D2 (in-house identifier: AfR46-P) [24]; anti-hCAP-D2 (in-house identifier: AfR51-5L) [32]; anti-hCAP-H (in-house identifier: AfR57-5L) [32]; anti-pT1339-hCAP-D2 (in-house identifier: AfR173-4P) [30]; anti-pT1384-hCAP-D2 (inhouse identifier: AfR175-4P) [30];…”

Section: Antibodiesmentioning

confidence: 99%

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Recombinant cyclin B-Cdk1-Suc1 capable of multi-site mitotic phosphorylationin vitro

Shintomi,

Masahara-Negishi,

Shima

et al. 2023

Preprint

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Cyclin-dependent kinase 1 (Cdk1) complexed with cyclin B phosphorylates multiple sites on hundreds of proteins during mitosis. However, it is not fully understood how multi-site mitotic phosphorylation by cyclin B-Cdk1 controls the structures and functions of individual substrates. Here we develop an easy-to-use protocol to express recombinant vertebrate cyclin B and Cdk1 in insect cells from a single baculovirus vector and to purify their complexes with excellent homogeneity. A series of in-vitro assays demonstrate that the recombinant cyclin B-Cdk1 can efficiently and specifically phosphorylate the SP and TP motifs in substrates. The addition of Suc1 (a Cks1 homolog in fission yeast) accelerates multi-site phosphorylation of an artificial substrate containing TP motifs. Importantly, we show that mitosis-specific multi-subunit and multi-site phosphorylation of the condensin I complex can be recapitulated in vitro using recombinant cyclin B-Cdk1-Suc1. The materials and protocols described here will pave the way for dissecting the biochemical basis of critical mitotic processes that accompany Cdk1-mediated large-scale phosphorylation.

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Section: Suc1 Accelerates M-cdk Phosphorylation Of a Substrate Contai...supporting

confidence: 78%

Section: Suc1 a Cdna Encoding Schizosaccharomyces Pombe Suc1 (Provide...mentioning

confidence: 99%

Section: Antibodiesmentioning

confidence: 99%

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Recombinant cyclin B-Cdk1-Suc1 capable of multi-site mitotic phosphorylationin vitro

Shintomi,

Masahara-Negishi,

Shima

et al. 2023

Preprint

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Quantitative imaging of loop extruders rebuilding interphase genome architecture after mitosis

Brunner,

Morero,

Zhang

et al. 2024

Preprint

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SummaryHow cells establish the interphase genome organization after mitosis is incompletely understood. Using quantitative and super-resolution microscopy, we show that the transition from a Condensin to a Cohesin-based genome organization occurs dynamically over two hours. While a significant fraction of Condensins remains chromatin-bound until early Gl, Cohesin-STAGl and its boundary factor CTCF are rapidly imported into daughter nuclei in telophase, immediately bind chromosomes as individual complexes and are sufficient to build the first interphase TAD structures. By contrast, the more abundant Cohesin-STAG2 accumulates on chromosomes only gradually later in Gl, is responsible for compaction inside TAD structures and forms paired complexes upon completed nuclear import. 0ur quantitative time-resolved mapping of mitotic and interphase loop extruders in single cells reveals that the nested loop architecture formed by sequential action of two Condensins in mitosis is seamlessly replaced by a less compact, but conceptually similar hierarchically nested loop architecture driven by sequential action of two Cohesins.

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Cell cycle-specific loading of condensin I is regulated by the N-terminal tail of its kleisin subunit

Cited by 2 publications

References 42 publications

Recombinant cyclin B-Cdk1-Suc1 capable of multi-site mitotic phosphorylationin vitro

Recombinant cyclin B-Cdk1-Suc1 capable of multi-site mitotic phosphorylationin vitro

Quantitative imaging of loop extruders rebuilding interphase genome architecture after mitosis

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